Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:20265407-20265506 100bp TCTGCATACTACCTCCTTAGGC TCCGAGGTTCTGGCTAAGAC
Mutant= 101 bp
Wild Type = 100 bp
WT Region: CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGTGTGTGTGTGTGTGCGTGATATTTTCCTAGCTATCAAGAGCATCTGCATACTACCTCCTTAGGCCCTGGCTTCCcactggctcattagagcacacacttcagtcaaccgctggccacttagtgtcttagccagaacctcggatccttcatgtccatttctgtgaacaggaatgatgctatctaagagacaggaaagcatcctcctgagagcttggtgacaggctgcactcagtaactgctgagcctgctgtccgtgcttcttccacaggtcatcctggaagcctgggtgaagggtgtgaaccccaaaggcaactccaccaatcccagcaactgggacttcgggagcagcttcttctttgcaggcacagtggtcaccaccataggtaagtgggagaggagtgaggcctcccttggccctgctgagcttggggtccattctaaatgccactgctggtctgggaatatgctttatccCGGAGGTGACAGACAGGGCACAGGTTGGGAAGTGGGTAGGTTGGCTGCAGCTGTAGTTGCCTGGACTCACTTCTCTGGGTCACACTGAAGGGTTAGAAAGAGCTATTCCTCAGTACA
Deleted Sequence cactggctcattagagcacacacttcagtcaaccgctggccacttagtgtcttagccagaacctcggatccttcatgtccatttctgtgaacaggaatgatgctatctaagagacaggaaagcatcctcctgagagcttggtgacaggctgcactcagtaactgctgagcctgctgtccgtgcttcttccacaggtcatcctggaagcctgggtgaagggtgtgaaccccaaaggcaactccaccaatcccagcaactgggacttcgggagcagcttcttctttgcaggcacagtggtcaccaccataggtaagtgggagaggagtgaggcctcccttggccctgctgagcttggggtccattctaaatgccactgctggtctgggaatatgctttatcc
400 bp deletion beginning at Chromosome 14 positive strand position 20,265,075 bp and ending after 20,265,474 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37333 | TCT GCA TAC TAC CTC CTT AGG C | Common | A | |||
37334 | TCC GAG GTT CTG GCT AAG AC | Wild type Reverse | A | |||
37335 | TGA GTC CAG GCA ACT ACA GC | Mutant Reverse | A | |||
37336 | Fluorophore-1 | CAG TCA ACC GCT GGC C | Quencher-1 | WT Probe | ||
37337 | Fluorophore-2 | AGA CAG GGC ACA GGT TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37333 | 0.40 uM |
37334 | 0.40 uM |
37335 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |